Consequences of the electrogenic function of the phagocytic NADPH oxidase

Abstract

NADPH oxidase of phagocytic cells transfers a single electron from intracellular NADPH to extracellular O2, producing superoxide (O.-2), the precursor to several other reactive oxygen species. The finding that a genetic defect of the enzyme causes chronic granulomatous disease (CGD), characterized by recurrent severe bacterial infections, linked O.-2 generation to destruction of potentially pathogenic micro-organisms. In this review, we focus on the consequences of the electrogenic functioning of NADPH oxidase. We show that enzyme activity depends on the possibilities for compensating charge movements. In resting neutrophils K+ conductance dominates, but upon activation the plasma membrane rapidly depolarizes beyond the opening threshold of voltage-gated H+ channels and H+ efflux becomes the major charge compensating factor. K+ release is likely to contribute to the killing of certain bacteria but complete elimination only occurs if O.-2 production can proceed at full capacity. Finally, the reversed membrane potential of activated neutrophils inhibits Ca2+ entry, thereby preventing overloading the cells with Ca2+. Absence of this limiting mechanism in CGD cells may contribute to the pathogenesis of the disease.

Figure 1

Possible mechanisms of charge compensation for electron transfer through the phagocytic NADPH oxidase (Phox).

Figure 2

Dependence of membrane depolarization (a) and K⁺(⁸⁶Rb) release (b) on the rate of ${\text{O}}_2^{·-}$ production. This figure was originally published by Rada, B. K., Geiszt, M., Káldi, K., Timár, Cs., & Ligeti, E. 2004 Dual role of phagocytic NADPH oxidase in bacterial killing. Blood 104, 2947–2953. © the American Society of Hematology.

Figure 3

Dependence of the proportion of charge compensation occurring by K⁺ release on the rate of ${\text{O}}_2^{·-}$ production (a) and correlation between membrane potential and transmembrane pH difference in activated eosinophilic granulocytes (b). In part b measurements were carried out either in the absence (open triangle) or in the presence (open square) of K⁺. Part (b) reproduced from The Journal of Experimental Medicine, 1999, 190, 183–194, by copyright permission of The Rockefeller University Press.

Figure 4

Relation of membrane depolarization and K⁺ release from PMA-activated neutrophils.

Figure 5

Relation of bacterial survival to the rate of ${\text{O}}_2^{·-}$ generation. Note: bacteria divide during the time of the experiment, explaining why in the case of severely impaired killing capacity, there are more bacteria at the end than at the start. This figure was originally published by Rada, B. K., Geiszt, M., Káldi, K., Timár, Cs., & Ligeti, E. 2004 Dual role of phagocytic NADPH oxidase in bacterial killing. Blood 104, 2947–2953. © the American Society of Hematology.

Figure 6

Relation of bacterial survival to membrane depolarization (a) or K⁺ release (b).

Figure 7

Comparison of the effect of 5 nM DPI and 10 μM ZnSO₄ on membrane depolarization and ${\text{O}}_2^{·-}$ generation (a) or bacterial survival (b).

Figure 8

Summary of the changes of K⁺ release (filled diamond), membrane depolarization (filled square) and bacterial survival (filled triangle) in the function of the rate of ${\text{O}}_2^{·-}$ generation.

Figure 9

Alteration of the Ca²⁺ signal in cells defective in ${\text{O}}_2^{·-}$ generation. The fMLP-induced Ca²⁺ signal was compared in differentiated PLB-985 myeloid cells containing an intact NADPH oxidase or no functioning enzyme (marked CGD) (a) and in peripheral blood neutrophils inhibited by 5 μM DPI (fMLP+DPI) or investigated in the absence of inhibitor (fMLP only) (b). This figure was originally published by Rada, B. K., Geiszt, M., van Bruggen, R., Német, K., Roos, D., & Ligeti, E. 2003 Clin. Exp. Immunol. 132, 53–60. © the British Society of Immunology.